Effect of Chlorhexidine Digluconate on Oral Bacteria Adhesion to Surfaces of Orthodontic Appliance Alloys
, Stjepan Spalj 2,5,* and Ivana Gobin 6Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsTitle: effect of chlorhexidine digluconate on oral bacteria adhesion to surfaces of orthodontic appliance alloys
The authors investigated the effect of chlorhexidine digluconate (CHX DG) mouthwash on the adhesion of oral bacteria to orthodontic appliances. They used four bacteria and two alloys in the study.
They found that CHX DG is effective at inhibiting bacterial adhesion to orthodontic elements, and they suggested to use for the chemical control of biofilms during orthodontic treatment with fixed appliances.
The study is interesting, it may be accepted to publish after minor revisions below.
- Page 3, line 5: ….haemin……. Is it “hemin” or “haemin”?
- How many replicates for each experiment is used? How many times of each experiment is repeated?
- The materials and methods section is not clear. I suggest to include a picture or diagram in the section that shows how the authors set up the experiments. The references should be added to the materials and methods section.
- What is the explanation of using the bags or candle for anaerobic condition? Why did they use different kind of anaerobic condition for each bacteria?
“…Streptococci were cultivated on Mutans-Sanguis agar (HIMEDIA, Mumbai, India) and incubated at 37° for 24 h with a candle to simulate anaerobic conditions. A. actinomycetemcomitans and V. parvula were cultivated on blood agar 95Appl. Sci. 2024, 14, x FOR PEER REVIEW 3 of 12 (Biolife, Milan, Italy) supplemented with 5% sheep blood (Biognost, Zagreb, Croatia) and incubated at 37° for 3-5 days in anaerobic conditions using AnaeroGen bags (GENbox anaerobe, bioMerieux SA, Marcy-l’Etolie, France)….”
Author Response
Reply in attach
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsEFFECT OF CHLORHEXIDINE DIGLUCONATE ON ORAL BACTERIA ADHESION TO SURFACES OF ORTHODONTIC APPLIANCE ALLOYS
The authors analysed the effect of chlorhexidine digluconate (CHX DG) mouthwash on the adhesion of oral bacteria to orthodontic appliances and conclude that CHX DG is effective at inhibiting bacterial adhesion to orthodontic elements and is recommended for the chemical control of biofilms during orthodontic treatment with fixed appliances.
Although it is not an original work, it is of interest because maintaining good hygiene during orthodontic treatments is important and any knowledge that improves it is important.
There are some topics that need attention:
2.1. Cultivation is not a word to use in a scientific work, it should be changed by inoculation or growth.
Change should be done in all document.
2.1. line 101 – Preparation of suspensions:
Line 103 – shouldn’t be 108 colonies?
Lines 103 and 105 – the exponent must be on top, it should be 107, for example.
2.2. line 107 – change the sentence, it is poorly constructed, it is not noticeable.
2.3. – “A 200 μL-mouthwash solution was applied to a microtitre plate that was diluted twice.” – Not clear! What was diluted twice? As it is written it seems that it is the plaque that is diluted!
“Because of the turbidity of the sample, it was not possible to determine the lowest concentration of the antimicrobial agent, the minimum inhibitory concentration (MIC).” – But after it is said “Samples from wells without turbidity were plated on Mutans-Sanguis and blood agar and incubated at 37° for 24–72 h.” – Confuse. Clarify.
2.6 . line 156 – “…was used to compare the adhesion between the four bacteria and the four periods.” – which periods are that? Clarify
Table 1 – The units must be the same, it makes no sense to compare percentages with concentrations. It must be harmonized. Either put everything in % or put it in concentration.
It is important to indicate the constituents of the commercial solutions used and whether they contain other substances with antimicrobial activity. It must be indicated.
The name of the microorganisms must be in italics. Review the entire document.
The hypotheses raised in the introduction (“The hypothesis was that CHX DG with the addition of alcohol would reduce adhesion to steel alloys, especially S. mutans bacteria”) is not discussed nor mentioned in the conclusion.
Little is explored and discussed about the fact that Perio Plus and Curasept products are more effective than CHX DG, about the difference in results.
The conclusion is very simplistic considering the results. Must be improved.
Comments on the Quality of English LanguageMinor editing, there are some typos in the writing
Author Response
Repy in attach
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript describes a study of the effect of chlorhexidine digluconate (CHX DG) mouthwash on the adhesion of oral bacteria to orthodontic appliances.
The topic is of practical value; the scope is rather narrow. The selection of only 1 commercial product for each alloy type limits the significance of the results in that it is known that the surface characteristics vary from different producers. Perhaps surface (morphology and/or roughness, hydropobillity) characterization is an easier remedy than including products from other main producers.
The manuscript lacks scientific rigor in that the hypothesis was not addressed later on. Further, the alcohol content should not be the main focus, thus the hypothesis needs to be re-formulated and addressed in Discussion, towards Conclusion. Please also double check some terms such as log10 (Fig. 1), Fig. 2 y-axis label, etc. All labels should start with capital letters.
Reproducibility is moderate because statistical analysis part is missing essential information such as confidence interval, alpha value, etc.
English usage needs to be improved to correct major mistakes of continuous misuse of the word adherence for adhesion, together with others.
Fig. 3 needs increased brightness.
Many statements are not referenced.
Statement in L280-281 needs to be rechecked and referenced. Alloy surface characteristics generally influence bacteria adhesion. “Both NiTi and SS exhibit hydrophilic characteristics” is this true? This needs references.
“In addition, NiTi has a higher roughness, polarity, and surface free energy than SS which makes NiTi more prone to bacterial adhesion.” Also needs references, suggest citing Biomedical NiTi and β-Ti Alloys: From Composition, Microstructure and Thermo-Mechanics to Application, Metals, 2022.
Comments on the Quality of English Languageextensive editing needed
Author Response
Reply in attach
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors did not addressed some points.
The references should be added to the materials and methods section.
Are the materials and methods that authors used for their study new methods? Did they use them first time in this study.
They may be modified from another study, so the references should be added.
Author Response
Dear Mr. Editor,
Thank you for the opportunity to revise our manuscript.
Please find reviewers’ concerns and out answers.
Sincerely
Stjepan Spalj, corresponding author
Reviewer 1.
The authors did not addressed some points. The references should be added to the materials and methods section. Are the materials and methods that authors used for their study new methods? Did they use them first time in this study? They may be modified from another study, so the references should be added.
Answer: The mentioned methods are regularly used by our group in research, apologies for the omission. We have now introduced the required references.
- Begić G, Petković Didović M, Lučić Blagojević S, Jelovica Badovinac I, Žigon J, Perčić M, et al. Adhesion of oral bacteria to commercial d-PTFE membranes: polymer microstructure makes a difference. Int. J. Mol. Sci. 2022, 23, 2983. doi: 10.3390/ijms23062983.
- Begić G, Badovinac IJ, Karleuša L, Kralik K, Cvijanovic Peloza O, Kuiš D, et al. Streptococcus salivarius as an important factor in dental biofilm homeostasis: influence on Streptococcus mutans and Aggregatibacter actinomycetemcomitans in mixed biofilm. Int. J. Mol. Sci. 2023, 24, 7249. doi: 10.3390/ijms24087249.
- Akca E, Akca G, Toksoy Topçu F, Macit E, Pikdöken L, Özgen S. The comparative evaluation of the antimicrobial effect of propolis with chlorhexidine against oral pathogens: an in vitro study. Biomed. Res. Int. 2016, 2016, 3627463. doi: 10.1155/2016/3627463.
Reviewer 2 Report
Comments and Suggestions for AuthorsEFFECT OF CHLORHEXIDINE DIGLUCONATE ON ORAL BACTERIA ADHESION TO SURFACES OF ORTHODONTIC APPLIANCE ALLOYS
I consider that the issues I raised have been corrected, however I think that the conclusion still deserves some investment. The hypothesis left in the introduction must be well answered in the conclusion and is not perfectly clear. The conclusion must be the answer to the hypothesis.
Author Response
Dear Mr. Editor,
Thank you for the opportunity to revise our manuscript.
Please find reviewers’ concerns and out answers.
Sincerely
Stjepan Spalj, corresponding author
Reviewer 2.
I consider that the issues I raised have been corrected, however I think that the conclusion still deserves some investment. The hypothesis left in the introduction must be well answered in the conclusion and is not perfectly clear. The conclusion must be the answer to the hypothesis.
Answer: Apologies for the mistake, we refined the research aim and removed the hypothesis that was redundant. We corrected the conclusion. Here is a new text:
“This study aimed to analyse the anti-adhesion effect of CHX DG, as a pure substance and commercially available CHX DG-based mouthwashes, on the adhesion of oral bacteria to SS and NiTi alloys, which are often used in orthodontic treatment. The hypothesis was that CHX DG would reduce adhesion to steel alloys, especially S. mutans bacteria, but not all commercial products in a similar manner.“
Reviewer 3 Report
Comments and Suggestions for AuthorsThe majority of the issues have been addressed.
The Conclusion needs to be further reformulated to comprehensively summarise the findings of the study and implications. CHX DG should be mentioned. "lower" and "better" are rather vague. They must be quantified and are they statistically significant?
Author Response
Dear Mr. Editor,
Thank you for the opportunity to revise our manuscript.
Please find reviewers’ concerns and out answers.
Sincerely
Stjepan Spalj, corresponding author
Reviewer 3.
The Conclusion needs to be further reformulated to comprehensively summarise the findings of the study and implications. CHX DG should be mentioned. "lower" and "better" are rather vague. They must be quantified and are they statistically significant?
Answer: We have corrected the conclusion as follows: ‘’Although there were differences between the strains and the tested agents, it can be concluded that Perio Plus most effectively inhibited the adhesion of all tested bacteria to the SS and NiTi alloys. A. actinomycetemcomitans was the most sensitive to all tested agents, while S. mutans showed the highest resistance. The effectiveness of the tested agents was better on NiTi alloys.”
